1. Field of the Invention
The present invention relates generally to continuous-flow, resistive-particle counting, and more particularly to stopped-flow aggregometry apparatus utilized in conjunction therewith in order to determine various parameters of blood platelets.
2. Description of the Prior Art
The following discussion refers to blood platelets. However, it must be emphasized that the principle of stopped-flow reactions is completely general. Any particles which can be detected by resistive-particle counters, and which react with other particles or chemicals, are potentially able to be analysed in the new apparatus.
Blood platelets play a key role in hemostasis, by providing various factors for the cascade pathway of coagulation, and in the direct formation of platelet plugs. They are also intimately involved in the important early events of atheroscherosis and in may bleeding disorders. In view of this, platelets having rightly attracted considerable attention towards understanding their formation in the bone marrow, their specific functions in the blood, and thei subsequent fate or destruction.
The development in the early 1960's of an optical, in-vitro test of platelet aggregation greatly helped to develop the fundamental biochemisty behind both normal and pathological platelet function. This test depends upon measuring the changes in the light transmittance of platelet-rich plasma (PRP) during aggregation wherein several characteristic phases occur. Initially, there is usually a small increase in the optical density for approximately 5-10 seconds after adding an aggregating agent, such as, for example, thrombin or adenosine diphosphate (ADP). This is referred to as the "shape change" phase and is associated with a disc-to-sphere transformation, during which time the platelets become covered with large numbers of projections or pseudopodia. Then, depending upon the concentration of the aggregating agent, the optical density of the PRP rapidly decreases as larger and larger platelet aggregates are formed. The second phase normally requires 1-2 minutes when ADP, adrenalin or thrombin are the aggregating agents, and an even longer time period, such as, for example, approximately 5 minutes, for collagen.
The optical tests therefore have an operational time scale of minutes, and it has proven difficult to obtain truly meaningful results concerning either the initial shape change or the rates of the extent of aggregation. Indeed, the theories of light scattering are complex, and the parameters of particle numbers volume and shape cannot be readily correlated with the changes in the optical density. These problems become particularly acute in view of a number of recent observations which show that many fundamental biochemical and structural events occur within seconds of initiating platelet aggregation.
By combining the same with continuous-flow techniques, successful techniques have been in clinical use for nearly 10 years, however, disadvantages nevertheless remain. In order to accomplish platelet counting, lysing agents must be used in order to remove the massive interference which would otherwise be caused by erythrocytes. For example, ammonium oxalate, in combination with a detergent, has been previously used, as has 2 M urea to lyse erythrocytes. A small disadvantage with such techniques, however, is that white cells are counted along with the platelets and have to be determined independently for a subsequent correction.
A major alternative to the optical sensing methods is resistive particle counting which has been a valuable analytical technique for the industrial, medical, and scientific communities for approximately 20 years, and which has provided information on particle numbers and particle size with respect to particles ranging in size from approximately 0.4-10.mu. or more in diameter. The more sophisticated apparatus have in fact been a considerable advance over earlier technology, providing up to seven parameters of clinical interest, mainly with regard to erythrocytes and white cells. In particular, clinical laboratories all over the world carry out enormous numbers of routine measurements of erythrocytes, white cell and blood platelets. Particle volumes are determined too, but this usually requires more ttime and expensive apparatus and is often not needed for medical diagnostic purposes. The advent of mini-computers has helped automate data reduction and has been very useful for calculating mean particle volumes and giving other statistical information from population histograms.
The major advantage of such apparatus is inherent in the original concepts where electrical information is available for determining both particle numbers and size, and potentially for shape too, with pulse-width analysis. The latter two parameters are of course not presently available with optical measuring systems. Other advantages are that pulse-height analysis is readily performed, and economical minicomputers provide capabilities of extensive data manipulation, aside from exhibiting great speed.
The technique of resistive counting is thus capable of providing the specific parameters of particle numbers and size, and thus has the potential of giving them during actual platelet aggregation. Several attempts have been made towards this end. For example, studies in the 1960's showed that ADP increased platelet volume by some 20 percent and it was considered that this reflected the disc-to-sphere transformation. Subsequently, independent techniques based on rapid centrifugation and the use of thrombocrits failed to reveal any increase in platelet volume. Therefore, the earlier resistive results were thought to stem from technical artifacts. An extensive recent investigation with the resistive technique has, however, shown that a highly-significant volume increase does occur in conjunction with the platelet shape change. This study took advantage of recent technical improvements in resistive counting and employed the basic conditions of the centrifugation procedures. It was found that both centrifugation and ethylenediaminetetraacetate (EDTA) caused platelets to swell and that EDTA also inhibited the change in volume. The net effect was to mask the true increase in platelet volume induced by ADP. Resistive counting has also been utilized to detect decreased amounts of single platelets during aggregation.
It seems clear then that resistive-particle counting can provide information on both platelet numbers and size during aggregation. However, several disadvantages inherent in the technique have prevented general application to platelet studies, particularly in terms of the very desirable goal of measuring initial rates of reaction. First, conventional resistive counting requires a sample of cells to be diluted some 100-1000 fold prior to analysis. This batch technique takes some 10-20 seconds before counting can be commenced. Secondly, at least 10 seconds of data accumulation are needed to obtain statistically-reliable numbers. Lastly, there has been no rapid way until very recently to measure particle volumes.
In addition, there are still other disadvantages of resistive counting. Simple clogging of the sensing orifice has been perhaps the major problem with all counters of such type, and one particular type of apparatus attempts to rectify such a problem through the provision of three orifice tubes each of white cells and erythrocytes, that is, six in all, with a corresponding duplication of circuitry. Comparison of the various data values from each then provides a means of detecting orifice blockage, however, the net result is complex and extremely expensive apparatus, which of course detracts from the inherent simplicity and ease of resistive-counter operation. Other approaches to the problem have also been utilized, such as, for example, a simple backflush plunger which is very valuable for cleaning nearly all blockages or the specific timing of the orifice flow rate has been another effective way to detect such problems. Still further, special redesign of the aperture tube, as disclosed in U.S. Pat. No. 3,746,976 to Hogg, wherein two orifices are used to minimize eddy currents, and therefore the generated electronic signals, has allegedly resulted in a self-cleaning orifice.
Other problems, however, have included poor resolution and skewed distributions of particles. The latter problem was apparently resolved, for example, with respect to erythrocytes, wherein the provision of longer orifice path lengths gave superior distibutions. Several recent approaches to the question of resolution demonstrate that it is often necessary to make complex modifications to the orifices. For example, Thom, as disclosed in U.S. Pat. No. 3,810,010, has designed a clever combination of two orifices, instead of one, wherein the particles are sucked out of the first orifice and surrounded by a sheath of particle-free electrolyte prior to their entry into the second sensing orifice. The improved resolution stems from the original concept of hydrodynamic focusing, however, Thom's apparatus is quite complex, and is dependent upon skilled glass-blowing and very precise geometries. In addition, due to the suction of the particles into the orifice together with the sheath of particle-free diluent, accurate counts of the detected particles are not obtained.
In U.S. Pat. No. 3,793,587 to Thom and Schulz, the apparatus disclosed therein serves to distinguish leucocytes from erythrocyte agglomerates, and to distinguish between two particles of equal volume but different shape. These goals are achieved by the use of two coaxial orifices of different path lenths, and a feed tube directed to the first orifice. As in U.S. Pat. No. 3,810,010, however, the same requires hydrodynamic sucking, which is a critical feature for success of the invention, and which, as noted hereinabove, draws in a quantity of particle-free diluent around the sucked particles through the first orifice. Consequently, the first orifice does not detect the true concentration of particles in the particle suspension.
Still further, a major disadvantage still remains in that intermittent and manual presentation of samples is still required. That is, individual liquid samples have to be handled and positioned over the sensing orifice prior to each analysis. In addition, relatively-large equantities of blood (1.3 ml) have to aspirate into the machine, mainly to flush away previous samples, and after appropriate dilution, the sample is subject to a static count. On the other hand, flow-counting techniques do have an inherent advantage over that static resistive approach in that they are more flexible in that multiple samples are able to be run in a continuous train towards the sensing zone. In addition, specific manipulations, such as reagent addition, are readily performed on the samples before analysis. Nevertheless, further improvement and/or refinement of such apparatus, and the technique associated therewith is desired.